Composition comprising hyaluronic acid and mepivacaine

ABSTRACT

A sterile composition for intraepidermal, intradermal, and/or subcutaneous administration including an effective amount of hyaluronic acid or a salt thereof, the effective amount of hyaluronic acid or salt thereof ranging from 1 to 3 wt % of the overall weight of the sterile composition, mepivacaine or a salt thereof as a first anesthetic agent at a concentration ranging from 0.05 to 3 wt % of an overall weight of the sterile composition, and a balanced salt solution in which the sterile composition exhibits greater stability over time and/or during heat treatment resulting in sterilization in comparison to the same composition having lidocaine as the anesthetic agent instead of mepivacaine.

FIELD OF INVENTION

The present invention relates to the field of sterile and injectablecompositions comprising hyaluronic acid or a salt thereof, andespecially soft tissue filler compositions, for in particular theaugmentation and/or repair of soft tissue, including periodontal tissue,and especially for treatment of defects and imperfections of keratinmaterials, like the skin. The invention is also directed to relatedmethods.

BACKGROUND OF THE INVENTION

There have been efforts to develop compositions useful to correctdefects in skin such as scars and wrinkles or to augment the tissue of asubject in order to improve the appearance of the skin, particularlyfacial skin. The principal method employed to correct such defectsinvolves injecting a filler composition into the dermal layer of theskin proximate to the effect or desired tissue augmentation.

The hyaluronic acid is the major component of the extracellular matrix(ECM). It is thus found in large quantities mainly in the skin. It isalso the major physiological component of the articular cartilage matrixand is particularly abundant in synovial fluid.

Accordingly, the hyaluronic acid, in its acid or salt form, is abiomaterial largely used as injectable filler material for tissueengineering application and especially for augmentation of dermal tissueor other soft tissue like gingival tissue.

Hyaluronic acid is a linear non-sulfated glycosaminoglycan composed ofrepeating units of D-glucuronic acid and N-acetyl-D-glucosamine (TammiR., Agren U M., Tuhkanen A L., Tammi M. Hyaluronan metabolism in skin.Progress in Histochemistry & Cytochemistry 29 (2): 1.-81, 1994).

In the skin, hyaluronic acid is primarily synthesized by dermalfibroblasts and epidermal keratinocytes (Tammi R., cited above). Throughits residues bearing a negative charge, hyaluronic acid acts as a waterpump for maintaining the elasticity of the skin.

In addition, the ECM is a dynamic structure with a structural andregulatory role for the tissues. The ECM is composed of collagen andelastin and also fundamental substance, mainly water, minerals andproteoglycans. This matrix gives to the skin its turgor and mechanicalproperties of firmness, elasticity and tone.

Regarding the skin, it is noticed that, with age, the amount ofhyaluronic acid and its degree of polymerization decreases, resulting ina decrease in the amount of water retained in the connective tissue.

Meanwhile, ECM components are degraded, mainly by endopeptidase typeenzymes called matrix metalloproteinases or MMPs.

Finally, decreases in cellular defenses increase damage and disordersinduced by external stresses such oxidative stress.

The skin is then subjected to an aging process leading to the appearanceof defects and blemishes of keratinous substances, in particular of theskin.

In the field of wrinkle fillers, gels consisting mainly of hyaluronicacid, the case arising chemically crosslinked, are injectedintradermally to fill the depression dug by the ride. The crosslinkingcan increase the persistence of the product within the dermis. Such gelsbased on hyaluronic acid, if necessary crosslinked, allow a reduction ofwrinkles by the mechanical effect of the filler resulting from thevacuum skin wrinkle.

However, it is known that the injection of such gels often produces apainful sensation for the patient, this sensation being furtherexacerbated more the gel is highly viscous and/or elastic.

Today, so as to overcome this technical problem, the main fillers basedon hyaluronic acid are available with a local anesthetic agent to ensuregreater patient comfort. This local anesthetic agent is only lidocaine,with a dosage of about 0.3%.

However, it is known that lidocaine may display the disadvantage,regarding its vasodilatory properties, to imply a too rapid absorptionby the patients body and sometimes an exacerbated occurrence of hematomawhich are, for obvious aesthetic reasons, to be avoided as much aspossible. A solution to overcome the above-mentioned problems mayconsist to implement the lidocaine in association with avasoconstrictor, in particular epinephrine (J. Endod. 2013 May;39(5):597-9). However, the presence of a vasoconstrictor likeepinephrine may require adding a preservative which isn't always inertwith respect to the patient and may conduct to allergies.

Therefore, it remains a need to develop gels consisting mainly ofhyaluronic acid, the case arising chemically crosslinked, whichovercomes the above-mentioned technical problems of the painfulsensation for the patient during the injection and which also do notinvolve any problem of allergies nor exacerbated occurrence of hematoma.

SUMMARY OF INVENTION

According to a first aspect, the invention relates to a methodcomprising at least the administering by injection to a patient in needthereof of:

(a) at least an efficient amount of hyaluronic acid or a salt thereof;and

(b) at least an efficient amount of mepivacaine or a salt thereof asanesthetic agent,

said mepivacaine being administered before or concurrently with thehyaluronic acid.

According to specific aspect, the method of the invention is intended tosoft tissue augmentation.

The target tissue may be skin but also gingival tissue.

More particularly, the present method may be efficiently used (i) forpreventing and/or treating the surface appearance of the skin.

The present method may thus advantageously be used (ii) to preventand/or treat cutaneous signs of chronological aging and/or induced byexternal factors such as stress, air pollution, tobacco or prolongedexposure to ultraviolet (UV) exposure, (iii) to prevent and/or treatimpaired surface appearance of the skin, (iv) to prevent and/or treatimpaired viscoelastic or biomechanical properties of the skin and/or (v)for the long-lasting filling of volume defects of the skin, and inparticular the filling of wrinkles.

Furthermore, the method of the invention may be advantageously also usedfor treating sites of deficiencies and defects in gingival architectureand contour and bony tissue such as occur with teeth loss, increase inage, periodontal disease and disorders, periodontal trauma and aftertooth implants, for the enhancement of fit and function of dentalprosthetics including but not limited to implants, crown, bridges . . ..

According to another embodiment, the method of the invention is alsouseful for cartilage regeneration.

According to another embodiment, the method of the invention may be usedas a treatment for arthritis.

At last, the hyaluronic acid is a thick transparent liquid similar tothe natural fluid located in eyes. Accordingly, it is already proposedfor maintaining eyes fluid volume during surgery. Thus the method of theinvention is also useful in most ophthalmic intraocular surgeries,including cataract extraction, Intraocular Lens (IOL) insertion andremoval, corneal surgery, glaucoma surgery, trauma surgery, ocularplastic surgery and muscle surgery.

According to a second aspect, the invention relates to a sterile andinjectable composition, more particularly a soft tissue fillercomposition, comprising an effective amount of at least hyaluronic acidor a salt thereof and including an effective amount of at leastmepivacaine or a salt thereof as anesthetic agent.

Finally, according to another aspect, the invention relates to a methodof preparing a sterile and injectable composition, more particularly asoft tissue filler composition, the method comprising at least the stepsof:

a) providing at least one gel of a hyaluronic acid or a salt thereof,said hyaluronic acid being selected under a crosslinked hyaluronic acidform, a non-crosslinked hyaluronic acid form or a mixture thereof;

b) adding to said gel of hyaluronic acid at least mepivacaine or a saltthereof as anesthetic agent; and

c) sterilizing the mixture obtained in step b).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

According to a first aspect, the invention relates to a methodcomprising at least the administering by injection to a patient in needthereof:

(a) at least an efficient amount of hyaluronic acid or a salt thereof;and

(b) at least an efficient amount of mepivacaine or a salt thereof asanesthetic agent,

said mepivacaine being administered before or concurrently with thehyaluronic acid.

The inventors have indeed found that a method according to the inventionmay constitute a viable alternative to current methods for in particularpreventing and/or treating the surface appearance of the skin whichconsider the implementation of hyaluronic acid with lidocaine but also(i) for treating sites of deficiencies and defects in gingivalarchitecture and contour and bony tissue, (ii) for ophthalmicintraocular surgeries, (ii) for the regeneration of the cartilage and(iv) in the treatment of arthritis.

Firstly, in contrast to lidocaine, mepivacaine do not displayssignificant vasodilatory properties than lidocaine.

Furthermore, mepivacaine has a pKa of 7.6 whereas lidocaine has a pKa of7.9.

Therefore, mepivacaine having a pKa lower than lidocaine, it displays abetter lipid solubility which improves its diffusion through lipidbarriers. Therefore, due to the fact that mepivacaine may be absorbedmore rapidly than lidocaine, mepivacaine is therefore appropriate tobetter prevent a painful sensation during the injection.

But above all, the inventors have unexpectedly observed than a fillercompositions according to the invention, i.e including mepivacaine asanesthetic agent, may be sterilized without significantly affecting thestability of the gel of hyaluronic acid.

It is indeed known than the gels of hyaluronic acid are particularlysensible to the heat treatment like required for their sterilization andthat this low stability may be increased by the presence of furthermaterials like the anesthetic agent. Unexpectedly, this undesirablephenomenon is significantly lowered with mepicavaine comparatively tolidocaine, as shown in the following examples 1 and 2. In addition, theinventors have shown that a composition according to the inventionfurther complies with the requirements in terms of stability in time, ashereinafter shown in example 3.

According to the inventors, the manifestation of these above-mentionedadvantageous effects within soft tissue filler compositions comprisinghyaluronic acid was not known.

Preferably, the hyaluronic acid or a salt thereof is administeredconcurrently with the mepivacaine or a salt thereof.

Preferably, the administering by injection is an intraepidermal and/orintradermal and/or subcutaneous injection. However it may be alsoadministered by a gingival, articular and intraocular road.

Hyaluronic Acid

As above-mentioned hyaluronic acid (also called hyaluronan orhyaluronate) is a linear non-sulfated glycosaminoglycan composed ofrepeating units of D-glucuronic acid and N-acetyl-D-glucosamine (TammiR., Agren U M., Tuhkanen A L., Tammi M. Hyaluronan metabolism in skin.Progress in Histochemistry & Cytochemistry 29 (2): 1.-81, 1994).

In the skin, hyaluronic acid is primarily synthesized by dermalfibroblasts and epidermal keratinocytes (Tammi R., cited above). Throughits residues bearing a negative charge, hyaluronic acid acts as a waterpump for maintaining the elasticity of the skin.

As above-mentioned, the present invention considers the implementationof hyaluronic acid as such but also a salt thereof.

Therefore, the hyaluronic acid in accordance with the invention may bemore particularly chosen from physiologically acceptable salts such asthe sodium salt, the potassium salt, the zinc salt, the silver salt andmixtures thereof, preferably the sodium salt.

Preferably, the hyaluronic acid according to the invention has a highaverage molecular weight, preferably ranging from 50 000 to 10 000 000Daltons, preferably from 500 000 to 4 000 000 daltons.

One particularly preferred salt of hyaluronic acid is sodium hyaluronate(NaHA).

As above-mentioned, hyaluronic acid is administered by injection in aneffective amount.

An “effective amount” of hyaluronic acid is an appropriate amount toobtain the desired technical effect, notably to have a visible result onthe surface appearance of the skin. In particular, an effective amountof hyaluronic acid is an appropriate amount of hyaluronic acid for agood filling of volume defects of the skin, and in particular thefilling of wrinkles.

Adjusting the amount of hyaluronic acid falls within the competence of aperson skilled in the art.

Advantageously, the hyaluronic acid or a salt thereof in a methodaccording to the invention is present in a solution.

In this regard, an effective amount of hyaluronic acid may range from0.1 to 5% by weight, preferably from 1 to 3% by weight, relative to thetotal weight of said solution comprising the hyaluronic acid or a saltthereof.

According to a particular embodiment, the hyaluronic acid may be presentunder an uncrosslinked form.

For the purpose of the present invention, the term “uncrosslinked” or“non-crosslinked” is understood in the context of the present inventionto mean a gel of hyaluronic acid which is not crosslinked or slightlycrosslinked, that is to say a gel having a phase-shift angle δ, measuredunder dynamic rheology conditions at 1 Hz, that is greater than 40° whensubjected to a stress above 1 Pa.

According to another particular embodiment, the hyaluronic acid may bepresent under a crosslinked form.

According to another particular embodiment, the hyaluronic acid may bepresent under crosslinked and non-crosslinked forms.

In this regard, the weight ratio “crosslinked hyaluronicacid/non-crosslinked hyaluronic acid” is preferably greater than 1.

According to a particular embodiment, the solution comprising thehyaluronic acid and being intended to be administered may comprise:

-   -   from 50% to 99% by weight, more preferably 70% to 95% by weight        of hyaluronic acid present in the form of a crosslinked gel,    -   from 1% to 50% by weight, preferably 5% to 30% by weight, of        hyaluronic acid present in the free form or a physiologically        acceptable salt thereof,

the ratio between the weight of the crosslinked hyaluronic acid gel andthe weight of the free hyaluronic acid being between 1:1 and 1:0.02.

When the hyaluronic acid is crosslinked, said crosslinked hyaluronicacid has preferably a degree of modification ranging from 0.1 to 20%,preferably from 0.4 to 10%.

By “degree of modification” is meant, in the sense of the presentinvention, the ratio between the number of moles of crosslinking agentattached to the hyaluronic acid and the number of moles of hyaluronicacid forming said crosslinked hyaluronic acid gel. This value may bemeasured by a NMR analysis 1D 1H of the crosslinked gel.

By “number of moles of hyaluronic acid” is meant the number of moles ofrepeating disaccharide units of the hyaluronic acid, a disaccharide unitbeing composed of D-glucuronic acid and D-N-acetylglucosamine linkedtogether by alternated beta-1,4 and beta-1,3 glycosidic bonds.

This degree of modification may be notably appreciated as disclosedherein after.

Thus, the characterizing of the degree of modification of the hyaluronicacid is carried out by spectroscopy by NMR (spectrometer Bruker Avance 1operating at 400 MHz (¹H)).

For BDDE, the degree of modification is obtained by integrating thesignal of ¹H NMR N-acetyl group (δ≈2 ppm) present in the hyaluronic acidand a signal present in the crosslinking agent (two —CH2- groups, δ≈1.6ppm). The ratio of integrals of these two signals (crosslinkingagent/NaHA) relates to the degree of modification, after correcting thenumber of protons attached to each signal.:

${{Degree}\mspace{14mu} {of}\mspace{14mu} {modification}} = \frac{\left\lbrack \frac{{Integral}\mspace{14mu} \delta_{H}1.6}{4} \right\rbrack}{\left\lbrack \frac{{Integral}\mspace{14mu} \delta_{H}2.0}{3} \right\rbrack}$

Crosslinking Agent

The term “crosslinking agent” is understood to mean any compound capableof inducing a crosslinking between the various chains of the hyaluronicacid.

The choice of this crosslinking agent clearly falls within thecompetence of a person skilled in the art.

A crosslinking agent in accordance with the invention may be chosen fromdifunctional or multifunctional epoxy crosslinking agents but alsopolyamines like for example hexamethylenediamine (HMDA) or endogeneouspolyamines.

By “endogenous polyamine” within the meaning of the present invention ismeant a polyamine naturally present in living organisms and particularlythe human body. As representative of endogenous polyamines, may beespecially mentioned those cited in eukaryotes animals, such as theputrescine (or 1,4-diaminobutane), the spermidine (or1,8-diamino-5-azaoctane) and the spermine(1,12-diamino-5,9-diazadodecane), preferably the spermine.

More particularly, a crosslinking agent according to the presentinvention may be preferably selected from the group consisting of1,4-butanediol diglycidyl ether (BDDE), 1,4-bis(2,3-epoxypropoxy)butane,1,4-bisglycidyloxybutane, 1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, an endogenous polyamine, andmixtures thereof.

Preferably, a crosslinking agent according to the invention is1,4-butanediol diglycidyl ether (BDDE).

In the particular embodiment wherein the crosslinking agent is apolyamine, and more particularly an endogenous polyamine, the couplingreaction with the hyaluronic acid may be carried out in the presence ofat least one activator, and the case arising associated with at leastone auxiliary coupling.

In this regard, the activator may be selected from water-solublecarbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC), 1-ethyl-3-(3-25 trimethylaminopropyl)carbodiimide hydrochloride(ETC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) and a saltthereof, and mixtures thereof, preferably is represented by the EDC.

Regarding the coupling auxiliary, when it is present, it may be selectedfrom N-hydroxy succinimide (NHS), N-hydroxybenzotriazole (HOBt),3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt),1-hydroxy-7-7azabenzotriazole (HAt) and N-hydroxysylfosuccinimide (sulfoNHS), and mixtures thereof, preferably is represented by the HOBt.

The crosslinking agent is implemented in an effective amount.

An “effective amount” of crosslinking agent is an appropriate amount toobtain an appropriate degree of crosslinking of hyaluronic acid.

Adjusting the amount of crosslinking agent falls within the competenceof a person skilled in the art.

Advantageously, an effective amount of crosslinking agent may range from0.05 to 15% by weight relative to the total weight of the hyaluronicacid or a salt thereof.

Advantageously, when the hyaluronic acid is crosslinked, and when thecrosslinking agent is BDDE, the amount in mole ratio of nBDDE/nhyaluronic acid may range between 0.01 and 0.5, preferably between 0.04and 0.25.

Anesthetic Agent

An anesthetic agent according to the present invention has theparticular advantage to reduce or eliminate the pain sensationexperienced by the patient during and/or following the injection.

Moreover, an anesthetic agent according to the present invention ischosen among compounds which do not raise risk of incompatibility withother compounds used in a composition according to the invention,especially with hyaluronic acid.

As above-mentioned, an anesthetic agent according to the presentinvention is mepivacaine, or a salt thereof.

The mepivacaine is a local anesthetic of the amino-amids family of theformula (I):

The mepivacaine is notably sold under the name Carbocaine® by thecompany Cook-Waite.

The mepivacaine is a molecule having a good stability. Its maindegradation product is 2,6-dimethylaniline. Mepivacaine is a moleculecontaining a chiral center (asymmetric carbon). Thus, there are the Rand S enantiomers. In the following examples, it is considered theracemic mixture of the two forms of mepivacaine.

Preferably, the mepivacaine is under a salt form, and more particularlythe salt of mepivacaine is chlorhydrate mepivacaine.

As above-mentioned, the mepivacaine or a salt thereof is administered byinjection in an effective amount.

An “effective amount” of mepivacaine or a salt thereof is an appropriateamount to effectively reduce or eliminate the pain sensation experiencedby the patient during and/or following the injection.

Adjusting the amount of mepivacaine or a salt thereof falls within thecompetence of a person skilled in the art.

Advantageously, the mepivacaine or a salt thereof in a method accordingto the invention is present in a solution.

In this regard, an effective amount of the mepivacaine or a salt thereofmay range from 0.05 to 3% by weight relative to the weight of thesolution comprising said mepivacaine or a salt thereof.

The method may further comprises the administration, the case arisingjoint to the mepivacaine, of at least one additional anesthetic agent ora salt thereof different from the mepivacaine.

This further anesthetic agent may be in particular selected fromambucaïne, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine,biphénamine, bupivacaine, butacaine, butamben, butanilicaine,butethamine, betoxycaïne, carticaïne, chloroprocaine, cocaethylene,cocaine, cyclométhycaïne, dibucaine, diméthysoquine, dimethocaine,diperodone, dycyclonine, ecgonidine, ecgonine, ethyl chloride,etidocaine, beta-eucaine, euprocine fenalcomine, formocaine, hexylcaine,hydroxytétracaïne, isobutyl p-aminobenzoate, leucinocaïne mesylate,levoxadrol, lidocaine, meprylcaïne, metabutoxycaïne, methyl chloride,myrtecaïne, naepaïne, octacaïne, orthocaïne, oxethazaine,parethoxycaine, phénacaïne, phenol, piperocaine, piridocaïne,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaïne, propoxycaine, pseudococaïne, pyrrocainc,ropivacainc, salicyl alcohol, tetracaine, tolycaïne, trimécïne,zolamine, or a salt thereof, and a mixture thereof.

Preferably, this additional anesthetic agent is lidocaine or a saltthereof.

According to a first embodiment, the additional anesthetic agent isadministered alone, previous to the hyaluronic acid and the mepivacaineor a salt thereof.

According to a second embodiment, the additional anesthetic agent isadministered together with the mepivacaine or a salt thereof, previousto the hyaluronic acid or a salt thereof.

According to a third embodiment, the additional anesthetic agent isadministered together with the mepivacaine or a salt thereof and thehyaluronic acid or a salt thereof.

In the specific embodiment where both anesthetic agents are contained ina same composition, they may be present therein in a ratio“mepivacaine/lidocaine” 1:1, preferably a ratio 1:0.1.

Balanced Salt Solution

According to a particular embodiment, the method according to theinvention may further comprise at least a step of administering byinjection to said patient of (c) at least a balanced salt solution,concurrently with said hyaluronic acid and/or mepivacaine, preferablywith mepivacaine.

The implementation of this balanced salt solution is particularlyinteresting in that it allows still reducing the injection pain (seeFarley J. S. et al., Regional Anesthesia A., 1994, Vol. 19: 48).

Preferably, said balanced salt solution is a phosphate buffered saline,and more particularly is a phosphate buffered saline and particularly aKH₂PO₄/K₂HPO₄ saline buffer.

According to a particular embodiment, said balanced salt solution mayfurther comprise at least one compound selected from the groupconsisting of an alpha-lipoic acid; N-Acétyl-L-cysteine; reducedglutathion; amino acid such as L-Arginine, L-Isoleucine, L-Leucine,monohydrated L-Lysine, Glycine, L-Valine, L-Threonine, L-Proline;pyridoxine Hydrochloride; dehydrated zinc acetate; pentahydrates coppersulphate, and mixture thereof.

Composition According to the Invention

As above-mentioned, the present invention also relates to a sterile andinjectable composition, in particular a soft filler composition,comprising an effective amount of at least hyaluronic acid or a saltthereof and including an effective amount of at least mepivacaine or asalt thereof as anesthetic agent.

By “sterile”, in the sense of the present invention, is meant anenvironment capable of guaranteeing to the considered compounds in acomposition according to the invention safety requirements for theadministration routes such as above-mentioned, notably into or throughthe skin. Indeed, for obvious reasons, it is essential that acomposition according to the invention be devoid of any contaminant bodycapable of initiating an undesirable side reaction at the host organism.

The hyaluronic acid, the crosslinking agent, and the anesthetic agent(s)are such as above-mentioned.

Regarding the hyaluronic acid, a composition according to the inventionmay comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weightof hyaluronic acid, relative to the total weight of said composition.

According to a particular embodiment, as above-mentioned, the hyaluronicacid may be present in crosslinked and non-crosslinked forms.

In this regard, the ratio “crosslinked hyaluronic acid/non-crosslinkedhyaluronic acid” is preferably greater than 1.

More particularly, a composition according to the invention maycomprise:

-   -   from 50% to 99% by weight, more preferably 70% to 95% by weight        of hyaluronic acid present in the form of a crosslinked gel,    -   from 1% to 50% by weight, preferably 5% to 30% by weight of        hyaluronic acid present in a non-crosslinked form,

the ratio between the weight of the crosslinked hyaluronic acid gel andthe weight of the non-crosslinked hyaluronic acid being between 1:1 and1:0.02.

Regarding the anesthetic agent(s), a composition according to theinvention may comprise from 0.01% to 5% by weight, preferably from 0.05%to 3% by weight of anesthetic agent(s), based on the total weight ofsaid composition.

By its injectable character, a composition according to the inventionnecessarily comprises a physiologically acceptable medium.

A “physiologically acceptable medium” means a medium devoid of toxicityand compatible with the injection and/or the application of thecomposition such as considered in the present invention.

The composition may comprise a solvent or a mixture of physiologicallyacceptable solvents.

The composition may comprise a physiologically acceptable aqueousmedium.

As an aqueous medium suitable for the invention, may be for examplementioned water.

As isotonic agents suitable for the preparation of a compositionaccording to the invention, it may be mentioned sugars and sodiumchloride.

According to a particular embodiment, a composition according to theinvention may further comprise at least a balanced salt solution such asabove-mentioned.

According to a particular embodiment, a composition according to theinvention may further comprise at least one compound selected from thegroup consisting of an alpha-lipoic acid; N-Acétyl-L-cysteine; reducedglutathion; amino acid such as L-Arginine, L-Isoleucine, L-Leucine,monohydrated L-Lysine, Glycine, L-Valine, L-Threonine, L-Proline;pyridoxine Hydrochloride; dehydrated zinc acetate; pentahydrates coppersulphate, and mixture thereof.

A composition according to the invention may comprise, in addition tothe above-mentioned compounds, at least one additional compoundcompatible with use in the field of soft tissue filler compositioncompositions.

In this regard, additional compounds which may be implemented in acomposition according to the invention may be chosen from copper salt,alpha-lipoic acid, acetylated derivative of cysteine or mixture thereof.

The amounts of these additional compounds depend on the nature of thecompound in question, the desired effect, and the destination of thecomposition according to the invention.

These parameters belong to the general skill of the art.

A composition further comprising at least one copper salt may preferablycomprises said copper salt in an amount ranging from 0.1 to 50 ppm basedon the total weight of said composition.

A composition further comprising at least one alpha-lipoic acidpreferably comprises said alpha-lipoic acid in an amount ranging from0.5 to 10,000 ppm, preferably from 5 to 100 ppm of alpha-lipoic acid,based on the total weight of said composition.

A composition further comprising at least one acetylated derivative ofcysteine preferably comprises said acetylated derivative of cysteine inan amount ranging from 0.5 to 10,000 ppm based on the total weight ofsaid composition.

Among the other additional compounds which may be used in the presentinvention, may be mentioned antioxidants, amino acids, vitamins,minerals, nucleic acids, co-enzymes, adrenal derivatives, and mixturesthereof, said additional compounds being distinct from those indicatedabove.

As an antioxidant, it may be mentioned glutathione, ellagic acid,spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols,flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, andmixture thereof.

As amino acid, there may be mentioned arginine, isoleucine, leucine,lysine, glycine, valine, threonine, proline, methionine, histidine,phenylalanine, tryptophan, and mixture thereof.

As vitamins and their derivatives, may be mentioned vitamins E, A, C, B,especially vitamins B6, B8, B4, B5, B9, B7, B12, and pyridoxine better.

As minerals, mention may in particular be made of zinc salts, magnesiumsalts, calcium salts, potassium salts, manganese salts, sodium salts,and mixtures thereof.

As nucleic acids, may be mentioned in particular the derivatives ofadenosine, cytidine, guanosine, thymidine, the cytodine and mixturethereof.

As co-enzymes, may be cited coenzyme Q10, CoA, NAD, NADP, and mixturesthereof.

As an adrenaline derivatives, may be mentioned adrenaline,noradrenaline.

In addition, a composition according to the invention may furthercomprise any excipient commonly used in the technical field, such as forexample mono- and/or di-hydrated dihydrogenophosphate sodium and sodiumchloride, in physiological concentrations.

The amounts of additional active agents and/or excipients of coursedepend on the nature of the compound in question, the desired effect,and the destination of the composition according to the invention.

According to a particular embodiment, a composition according to theinvention may have a complex viscosity η* of between about 5 Pa*s andabout 450 Pa*s when measured at about 1 Hz.

According to a particular embodiment, a composition according to theinvention made up of crosslinked hyaluronic acid may have a viscositybetween 200 and 2000 Pa·s, preferably between 1000 and 1800 Pa·s.

The viscoelastic properties of a composition according to the inventionmay be measured using a rheometer (notably Haake RS6000) with acone/plate geometry (1° cone angle/35 mm diameter plate). A strain scanis carried out and the elastic modulus G′ (in Pa) and the phase-shiftangle δ (in°) are measured for a stress of 5 Pa.

Method for Preparing a Sterile and Injectable Composition According tothe Invention

As above-mentioned, the present invention also relates to a method ofpreparing a sterile and injectable composition, more particularly a softtissue filler composition, the method comprising at least the steps of:

a) providing at least one gel of a hyaluronic acid or a salt thereof,said hyaluronic acid being selected under a crosslinked hyaluronic acidform, a non-crosslinked hyaluronic acid form or a mixture thereof;

b) adding to said gel of hyaluronic acid at least mepivacaine or a saltthereof as anesthetic agent; and

c) sterilizing the mixture obtained in step b).

According to a preferred embodiment, the mepivacaine or a salt thereofis added to an injectable form of hyaluronic acid.

In other words, the gel of hyaluronic acid considered in step a) alreadyexhibits a concentration in hyaluronic acid, in crosslinking agentresidues if presents, and physiological and/or pH conditions which arecompatible with an injectable use, in particular in the fields of theapplications considered above.

By this way, the risks affecting the stability of the hyaluronic acid inthe mixture hyaluronic acid and mepivacaine by imposing additionalpurification steps, such as above-defined, are significantly reduced.

The mixture advantageously undergoes only one sterilization step. Thisstep is preferably carried out on the mixture already packaged in itsdelivery device, usually a syringe, as herein after defined.

Advantageously, the sterilization step can be performed by thermalmeans.

Advantageously, the sterilization is carried out at a temperatureranging from 120 to 140° C.

In particular, the sterilization step can be performed in an autoclave(moist heat) T° C.≥121° C., to obtain a Fθ>15 (sterilizing value).

In this regard, and as above-mentioned, a composition according to theinvention is particularly advantageous in that it displays a veryinteresting high resistance to this sterilization step.

Indeed, as shown in the examples 1 and 2, the loss of G′ generated bysterilization for a composition according to the invention is less thanfor a composition comprising lidocaine instead of mepivacaine.

According to a particular embodiment, the method for preparing a gel ofhyaluronic acid according to the invention may further comprise a stepd) consisting of adding an additional anesthetic agent or a saltthereof, different from the mepivacaine or a salt thereof, such asabove-mentioned, preferably lidocaine or a salt thereof, said step d)being carried out before, simultaneously and/or after the step b).

When the gel considered in step a) comprises a crosslinked hyaluronicacid form, this last may be obtained beforehand from an uncrosslinkedform of hyaluronic acid.

The reticulation may be performed by a conventional way with at leastone crosslinking agent, such above-mentioned.

The aqueous mixture comprising the crosslinking agent and the hyaluronicacid to crosslink is advantageously homogenized before performing thecrosslinking.

The purpose of this operation is more particularly for the hyaluronicacid or a salt thereof in the aqueous medium to be hydrated andhomogenized perfectly and thus to help to optimize the properties of thegel of hyaluronic acid expected. This step of homogenization is moreimportant when the hyaluronic acid has a high molecular weight, becausethe hydration of such a compound tends to result in the formation of ahigh-viscosity solution within which the appearance of agglomerates iscommonly observed.

The purpose of this operation is also intended to homogenize perfectlythe crosslinking agent within the mixture so as to subsequently assure ahomogeneous crosslinking reaction.

The homogenization is considered to be satisfactory when the solutionobtained is uniformly coloured, with no agglomerates, and has a uniformviscosity. The homogenization may advantageously be carried out undermild operating conditions so as to prevent degradation of the hyaluronicacid chains.

The duration of this homogenization step depends on the nature of thehyaluronic acid or a salt thereof, and more particularly the molecularweight and the concentration thereof, on the operation conditions withinthe aqueous medium and on the homogenizing device used, generally adevice which allows a soft mechanical agitation.

Preferably, a homogenization step may take place over a time of lessthan 200 minutes, preferably less than 150 minutes, or even between 15and 100 minutes.

The purpose of the crosslinking reaction is to create bridges betweenthe hyaluronic acid chains making it possible to obtain a dense solidthree-dimensional network from a viscous solution.

The particular conditions to be adopted in order to stimulate thecrosslinking reaction may depend on the molecular weight of thehyaluronic acid, on the aqueous medium and on the nature of thecrosslinking agent. In general, this reticulation may be achieved bybringing the mixture comprising the non crosslinked hyaluronic acid andat least one crosslinking agent as above-mentioned, into contact with atriggering element, or stimulant, such as, for example by heating orexposure to UV, or even by bringing said mixture into contact with amaterial of the catalyst type.

The choice of such a triggering element falls within the generalknowledge of a person skilled in the art.

In the context of the present invention, this triggering element isadvantageously represented by an increase in temperature imposed on themixture “non-crosslinked hyaluronic acid/aqueous medium/crosslinkingagent”.

A particularly suitable temperature for the crosslinking reaction isbetween 35° C. and 60° C., preferably between 45 and 55° C., and betterstill between 48 and 52° C.

The degree of crosslinking also depends on the crosslinking time and ofthe temperature imposed on the mixture “non-crosslinked hyaluronicacid/aqueous medium/crosslinking agent”. The longer the time is, thehigher the degree of crosslinking will be, with however an optimum notto be exceeded without running the risk of degrading the hyaluronicacid.

Thus, at a temperature ranging from 35° C. and 60° C., the crosslinkingreaction may be carried out over a time ranging from 30 to 300 minutes,preferably 100 to 200 minutes, and better still 150 to 190 minutes.

Preferably, the crosslinking conditions are adjusted to obtain a degreeof crosslinking such that the gel of crosslinked hyaluronic acid formedis a viscous, viscoelastic gel, or even a solid gel.

Stopping the crosslinking reaction requires exposing the crosslinked gelor, during crosslinking, even the receptacle containing it, toconditions propitious for stopping the crosslinking or else toconditions capable of stopping the formation of bonds between thevarious hyaluronic acid chains.

For example, with regard to the thermal conditions that will be appliedto stimulate the crosslinking process, the crosslinking may be stopped:

-   -   by simply removing the receptacle from the thermostatted bath        and cooling it until it returns to room temperature;    -   by placing the receptacle in a bath of cold water, preferably at        a temperature below room temperature, until the temperature        within said receptacle is close to room temperature; or even    -   by extracting the gel from said receptacle.

According to a particular embodiment, the homogenization andcrosslinking may be carried out within a hermetic cavity delimited atleast partially by a deformable wall, preferably made within adeformable pouch, which may be deformed at least manually by palpation,such as described notably in WO 2010/131175.

The gel obtained at the end of the crosslinking step cannot in generalbe injected directly, in particular because of its high hyaluronic acidconcentration and/or of the possible presence of crosslinking agentresidues or else because of its physiological and/or pH conditionsincompatible with use in the fields of applications considered above.

Furthermore, some gels may especially have too high a stiffness to beinjected as such into a patient. Therefore, several additional steps,known to those skilled in the art, can be carried out to obtain aninjectable hydrogel. More particularly, a step of neutralizing andexpanding this gel is required in order to give it its implantqualities. The chains of the hyaluronic acid network are then stretchedand hydrated, while the pH is brought to that of the dermis.

A step of protecting and redensifying the gel can also be carried outfor further improving the qualities of the implant, according to theknow-how of a person skilled in the art. The gel must be physiologicallyformulated by virtue of the presence of salts in equivalent amounts tothose of the medium injected.

For even higher purity, an additional purification step may also becarried out.

Advantageously, the mepivacaine or salt thereof and, the case arising,additional anesthetic agent(s), is/are added at the end of this protocolof preparation of an injectable form of hyaluronic acid, just before thestep of sterilization, such as above-defined.

According to a particular embodiment, the hydrogel added of mepicacaineand optionally of additional anesthetic agent(s), may be used to fillsyringes under controlled atmosphere conditions, said syringes thenpossibly undergoing a sterilization step, preferably a thermalsterilization step such as above-defined.

Administration of the Composition

A composition according to the invention can be injected using any ofthe known methods in the art.

Particularly, a composition of the invention may be administered bymeans of an injection device suitable for intraepidermal and/orintradermally and/or subcutaneously.

A composition of the invention may also be administered by means of aninjection device suitable for gingival, articular and/or intraocularregions.

The injection device, notably when a composition of the invention isadministered by means of an injection device suitable for intraepidermaland/or intradermally and/or subcutaneously, may be selected from asyringe, a set of microsyringes, a laser device, a hydraulic device, aninjection gun, a needleless injection device, a rolling withmicroneedles.

Preferably, the injection device may be selected from a syringe or a setof microsyringe.

In an alternative embodiment, the injection device can be adapted to thetechnique of mesotherapy.

Mesotherapy is a treatment technique by intraepidermal and/orintradermally and/or subcutaneously active(s) product(s).

The administration intraepidermal and/or intradermally and/orsubcutaneously according to the invention is to inject a composition ofthe invention in an epidermal region, dermo-epidermal and/or dermal.

The injection device may comprise any conventionally used injection suchas hypodermic needle or cannula.

A needle or a cannula according to the invention can have a diameterranging between 18 and 34 G, preferably 25 to 32 G, and a length varyingfrom 4 to 70 mm, preferably 4 to 25 mm.

The needle or cannula is preferably disposable.

Advantageously, the needle or cannula is associated with a syringe orother device capable of delivering through the needle or cannula saidinjectable composition.

According to one embodiment, a catheter may be inserted between theneedle/cannula and syringe.

In known manner, the syringe can be operated manually by thepractitioner or by a syringe holder as guns.

Throughout the description, including the claims, the expression“comprising a” should be understood as being synonymous with “comprisingat least one” unless specifically stated otherwise.

The expressions “between . . . and . . . ” and “ranging from . . . to .. . ” should be understood to mean that the limits are inclusive, unlessspecified otherwise.

The following examples and figures are presented by way of non-limitingillustration of the invention.

EXAMPLES

Material and Method:

The protocol herein after defined regarding the measure of theviscoelastic properties of a composition apply for the followingexamples.

The viscoelastic properties of a composition are measured using arheometer (Haake RS6000) with a cone/plate geometry (1° cone angle/35 mmdiameter plate). A strain scan is carried out and the elastic modulus G′(in Pa) and the phase-shift angle δ (in°) are measured for a stress of 5Pa.

Example 1: Protocol of Preparing a Composition According to theInvention Implementing a Non Crosslinked Hyaluronic Acid and AnalysisRegarding its Stability to the Sterilization

3 g of hyaluronic acid (1.5 MDa,) and 197 g of a phosphate buffer (asdefined in FR 2 979 539) are mixed.

The protocol of preparing is as follows:

1. tridimensional homogenization of the mixture “hyaluronicacid+phosphate buffer” at room temperature in an airtight container(Nalgene jar) for a minimum of 20 hours;2. separating the obtained non-crosslinked gel into 2 equivalentfractions (called hereinafter fractions A1 and A2);3. regarding fraction A1, adding to gel 1% by weight of a solution ofmepivacaine (30% by weight in phosphate buffer medium) and 0.4% byweight of a solution of NaOH 1%; or4. regarding fraction A2, adding to gel 1% by weight of a solution oflidocaine (30% by weight in phosphate buffer medium) and 0.4% by weightof a solution of NaOH 1%; and5. homogenization, packaging in syringes 1 mL for injection, andautoclaving (F0>15).

Results:

Tables below gives the values of the elastic moduli G′ (in Pa) of thehydrogels obtained and of the extrusion force F(N).

F(N) at 12.5 mm/min Dynamic oscillatory rheology Ser Schott 1 mL F = 5Hz F = 1 Hz Needle TSK HPC 30 Fraction A1 G′ (Pa) G′ (Pa) G: 120347Mepivacaine Non-sterilized 103.3 36.3 9 0.3% Sterilized  45.0  9.6 9Loss on sterilization  56.4 73.5 (in %)

The loss in sterilization for G′ at F=5 Hz is calculated as follows:(103.3−45.0)/103.3=56.4 (en %)

F(N) at 12.5 mm/min Dynamic oscillatory rheology Ser Schott 1 mL F = 5Hz F = 1 Hz Needle TSK HPC 30 Fraction A2 G′ (Pa) G′ (Pa) G: 120347Lidocaine Non-sterilized 105.9 37.4 9 0.3% Sterilized  36.7  7.0 9 Losson sterilization  65.4 81.2 (in %)

In view of the above, it therefore appears that the stability to thesterilization step of a composition comprising uncrosslinked hyaluronicacid is at least equivalent with mepivacaine than with lidocaine.

The results are even significantly better with mepivacaine.

Example 2: Protocol of Preparing a Composition According to theInvention Implementing Crosslinked and Non Crosslinked Hyaluronic Acidand Analysis Regarding its Stability to the Sterilization

8 g of hyaluronic acid (4 MDa), 58.7 g of sodium hydroxide (NaOH) 1% and0.6 g of butanediol diglycidyl ether (BDDE) are implemented. Thecrosslinking reaction is induced by an incubation for 3 hours at 52° C.

The protocol of preparing is as follows:

1. Homogenization of the mixture “hyaluronic acid+NaOH 1%” at roomtemperature in an airtight and deformable container (pouch such asconsidered in the patent application WO 2010/131175) during about 1H30so as to obtain a perfectly homogeneous viscous solution;2. Adding the crosslinking agent (BDDE) into the receptacle and newhomogenization at room temperature for about 20 min;3. Incubation for 3 hours at 52° C. of the receptacle containing theviscous solution of “hyaluronic acid/sodium hydroxide 1%/BDDE” so as toinitiate the crosslinking step;4. Neutralization, swelling and homogenizing the obtained solid(solution of crosslinked hyaluronic acid) in a phosphate buffer (asdefined in FR 2 979 539), and adding a non-crosslinked gel containing 4g of HA 4 MDa and 668 g of supplemented phosphate buffer, so as toobtain a hydrogel having 11 mg/g in hyaluronic acid, at pH aroundneutrality;5. Purification by dialysis during 48 hours, and then collecting thepurified gel in a tank homogenization;6. Sieving of the whole gel (230 μm);7. Separation of the collected gel into two fractions (Fraction B1 andB2);8. Incorporation to the gel of 1% by weight of a solution of mepivacaine(30% by weight in medium phosphate buffer) and of 0.4% by weight of asolution of NaOH 1% (=Fraction B1);9. Incorporation to the gel of 1% by weight of a solution of lidocaine(30% by weight in medium phosphate buffer) and of 0.4% by weight of asolution of NaOH 1% (=Fraction B2);10. Homogenization, packaging in syringes 1 mL for injection andsterilization.

Results:

Tables below gives the values of the elastic modulus G′ (in Pa) of thehydrogels obtained and of the extrusion force F(N).

Dynamic F(N) at 12.5 oscillatory mm/min rheology Ser Schott 1 mL F = 1Hz Needle TSK HPC 30 Fraction B1 G′ (Pa) G: 120347 MepivacaineNon-sterilized 20.4 10 0.3% Sterilized  8.1 10 Loss on sterilization60.3 (in %)

Dynamic F(N) at 12.5 oscillatory mm/min rheology Ser Schott 1 mL F = 1Hz Needle TSK HPC 30 Fraction B2 G′ (Pa) G: 120347 Lidocaine 0.3Non-sterilized 19.2 10 0.3% Sterilized  6.2 10 Loss on sterilization67.6 (in %)

The loss in sterilization is calculated as displayed in example 1.

In view of the above, it therefore appears that the stability to thesterilization step of a composition comprising crosslinked anduncrosslinked hyaluronic acid is at least equivalent with mepivacainethan with lidocaine.

The results are even better with mepivacaine.

Example 3: Protocol of Preparing a Composition According to theInvention Implementing Crosslinked and Non Crosslinked Hyaluronic Acidand Analysis Regarding its Stability in Time

10 g of hyaluronic acid (1.5 MDa,) 73 g of sodium hydroxide 1% and 0.9 gof butanediol diglycidyl ether (BDDE) are mixed. Then, the crosslinkingreaction is induced by incubation for 3 hours at 52° C.

The preparation procedure is as follows:

1. homogenizing the mixture at room temperature “hyaluronic acid+sodiumhydroxide 1%, in an airtight and deformable container (pocket) for about1H30 to obtain a perfectly homogeneous viscous solution;2. adding the crosslinking agent (BDDE) into the container and furtherhomogenization at room temperature for about 20 min;3. incubation for 3 hours at 52° C. of the receptacle containing theviscous solution of hyaluronic acid/sodium hydroxide 1%/BDDE so as toinitiate the crosslinking step;4. neutralization, swelling and homogenizing the obtained solid(solution of crosslinked hyaluronic acid) in a phosphate buffercontaining 2 g of non-crosslinked hyaluronic acid (1.5 MDa), so as toobtain a hydrogel at 25 mg/g of hyaluronic acid at pH close toneutrality;5. purification by dialysis (during 48 hours), and then collecting thepurified gel in a degassing vessel/homogenization;6. incorporation in the gel of 1% by weight of a solution of mepivacaine(30% by weight in a phosphate buffer medium) and of 0.4% by weight of aNaOH solution 1%;7. homogenization, degassing, packaging in syringes 1 mL for injectionand sterilization (F0>15).

Results:

Table below gives the values of the phase-shift angle δ (°) and theextrusion force.

Rheology amplitode Extrusion Force, scan (Cone/plate) measured with δ(°) measures needle TSK 30G1/2 Analysis at 5 Pa and 1 Hz F (N), at 12.5mm/min pH T0 17.8 ± 2.0 24.2 ± 2.5 7.1 T0 + 19 months 19.1 ± 2.0 23.1 ±2.5 7.1

The results obtained 19 months after the date of manufacture of the gelare equivalent to those obtained initially, and thus demonstrate thestability of the gel.

The disclosure of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

The disclosure of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A sterile composition for intraepidermal,intradermal, and/or subcutaneous administration comprising: an effectiveamount of hyaluronic acid or a salt thereof, the effective amount ofhyaluronic acid or salt thereof ranging from 1 to 3 wt % of the overallweight of the sterile composition, mepivacaine or a salt thereof as afirst anesthetic agent at a concentration ranging from 0.05 to 3 wt % ofan overall weight of the sterile, injectable composition, and a balancedsalt solution, wherein the sterile composition exhibits greaterstability over time and/or during heat treatment resulting insterilization in comparison to the same composition having lidocaine asthe anesthetic agent instead of mepivacaine.
 2. The sterile compositionaccording to claim 1, wherein the hyaluronic acid comprises crosslinkedhyaluronic acids, non-crosslinked hyaluronic acids, or a mixturethereof.
 3. The sterile composition according to claim 2, wherein thesalt of mepivacaine is chlorhydrate mepivacaine.
 4. The sterilecomposition according to claim 1, further comprising a second anestheticagent or a salt thereof as a second anesthetic agent, the secondanesthetic agent comprises at least one of ambucaine, amolanone,amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethysoquine, dimethocaine, diperodone,dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocine fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, meprylcaine, metabutoxycaine, methyl chloride,myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycane, trimecaine,zolamine, or a salt thereof, or mixtures thereof.
 5. The sterilecomposition according to claim 1, wherein the balanced salt solution isa phosphate saline buffer.
 6. The sterile composition according to claim1, wherein the hyaluronic acid has an average molecular weight rangingfrom 50,000 to 10,000,000 Daltons.
 7. The sterile composition accordingto claim 1 wherein the hyaluronic acid comprises crosslinked andnon-crosslinked hyaluronic acid at a ratio of 1:1 to 1:0.02 ofcrosslinked to non-crosslinked hyaluronic acid in the single, sterileinjectable composition.
 8. The sterile composition according to claim 7,wherein mepivacaine or salt thereof is present at a concentration of 0.3wt % of the overall weight of the single, sterile injectablecomposition, and the hyaluronic acid or the salt thereof comprisescrosslinked hyaluronic acids.
 9. The sterile composition according toclaim 1, further comprising an additional compound comprising at leastone of an antioxidant(s), amino acid(s), vitamin(s), mineral(s), nucleicacid(s), co-enzyme(s), adrenaline, noradrenaline, or mixtures thereof.10. The sterile composition according to claim 9, wherein the additionalcompound is an antioxidant.
 11. The sterile composition according toclaim 10, wherein the antioxidant comprises glutathione, ellagic acid,spermine, resveratrol, retinol, L-carnitine, polyol(s), polyphenol(s),flavonol(s), theaflavine(s), catechin(s), caffeine, ubiquinol,ubiquinone, or mixtures thereof.
 12. The sterile composition accordingto claim 10, wherein the antioxidant is at least one polyol.
 13. Thesterile composition according to claim 9, wherein the additionalcompound comprises an antioxidant and an amino acid, the amino acidselected from the group consisting of arginine, isoleucine, leucine,lysine, glycine, valine, threonine, proline, methionine, histidine,phenylalanine, tryptophan, or mixtures thereof.
 14. The sterilecomposition according to claim 9, wherein the additional compoundcomprises an antioxidant and a vitamin, the vitamin selected from thegroup consisting of vitamin E, vitamin A, vitamin C, vitamin B, ormixtures thereof.
 15. The sterile composition according to claim 9,wherein the additional compound comprises an antioxidant and a mineral,the mineral selected from the group consisting of zinc salts, magnesiumsalts, calcium salts, potassium salts, manganese salts, sodium salts, ormixtures thereof.
 16. The sterile composition according to claim 9,wherein the additional compound comprises an antioxidant and a nucleicacid, the nucleic acid selected from the group consisting of adenosine,cytidine, guanosine, thymidine, and mixtures thereof.
 17. The sterilecomposition according to claim 9, wherein the additional compoundcomprises an antioxidant and a co-enzyme, the co-enzyme selected fromthe group consisting of coenzyme Q10, CoA, NAD, NADP, or mixturesthereof.
 18. The sterile composition according to claim 1, wherein thehyaluronic acid or a salt thereof comprises hyaluronic acid crosslinkedwith at least one crosslinker selected from the group consisting of1,4-butanediol diglycidyl ether (BDDE), 1,4-bis(2,3-epoxypropoxy)butane,1,4-bisglycidyloxybutane, 1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, an endogenous polyamine, andmixtures thereof.
 19. The sterile composition according to claim 1,wherein the hyaluronic acid or a salt thereof comprises hyaluronic acidcrosslinked with 1,4-butanediol diglycidyl ether (BDDE).